Mass Spectrometric Detection of Drug-Resistant HIV

描述(申请人提供)：美国约有100万人感染HIV-1，每年新增4万例。虽然大多数患者从高效抗逆转录病毒疗法(HAART)中受益匪浅，但他们往往会出现与病毒突变相关的多重耐药性，这给长期治疗带来了问题。了解HIV-1逆转录病毒变异谱对于有效地计划这些患者的药物治疗是极其重要的。然而，测序昂贵、耗时，而且不能检测到低水平的耐药菌株。 这项建议的主要目标是开发一种高灵敏度、高成本效益和高通量的系统，用于基于免费蛋白质表达和质谱仪检测HIV-1病毒耐药株。这种方法是基于这样一个事实，即艾滋病毒中的耐药突变会改变编码蛋白质的质量。在第一阶段，将使用药物靶向病毒逆转录酶和蛋白酶的野生型(HXB2)和突变序列对整体方法进行广泛评估。利用RT-PCR扩增的DNA模板，来自每种蛋白质中可能包含抗药性突变的区域的多肽将在兔网织红细胞或大肠杆菌的无细胞提取物中表达。为了减少非细胞表达多肽的蛋白降解，初步研究发现了一个基于未修饰的大肠杆菌翻译因子的高保真重组翻译系统。在AmberGen早期工作的基础上，将对几种从无细胞提取物中快速分离多肽片段的方法进行评估，包括：i)N末端表位掺入，ii)tRNA介导的蛋白质工程(TRAMPE)和iii)光解生物素。对纯化的HIV多肽进行MALDI-TOF质谱分析，包括对单个多肽和通过多重聚合酶链式反应和所有下游步骤产生的多个多肽进行分析。结果将与包括DNA测序在内的其他艾滋病毒逆转录病毒基因分型方法进行比较。在第二阶段，将根据行业标准96孔或384孔样本格式开发用于HIV-1耐药性测试的高通量MS系统。国际公认的艾滋病毒-1耐药性专家理查德·达奎拉博士将担任该项目的顾问和样本来源。传染病专家雷蒙德·特利耶博士也将担任顾问，他曾使用质谱仪来表征丙型肝炎病毒的准物种变异。

DESCRIPTION (provided by applicant): There are approximately one million persons in the U.S. infected with HIV-1, with 40,000 new cases reported each year. While most of these patients greatly benefit from highly active antiretroviral therapy (HAART), they often develop multi-drug resistance associated with viral mutations, which causes problems for long-term treatment. Knowledge of the spectrum of HIV-1 retroviral mutations is extremely important in order to effectively plan drug treatment for these patients. However sequencing is expensive, time-consuming and does not detect low-levels of drug-resistant strains. The principal objective of this proposal is to develop a highly sensitive, cost-effective and high throughput system for detecting drug-resistant strains of HIV-1 virus based on call-free protein expression and mass spectrometry. This approach is based on the fact that drug resistant inducing mutations in HIV alter the mass of the encoded proteins. During Phase I, the overall approach will be extensively evaluated using wild type(HXB2) and mutant sequences of the drug targeted viral reverse transcriptase and protease. Peptides from regions of each protein likely to contain drug-resistant mutations will be expressed in cell-free extracts from rabbit reticulocyte or E. coli using RT-PCR amplified DNA templates. In order to reduce proteolyUc degradation of cell-free expressed peptides, ia problem revealed in preliminary studies, a high fidelity reconstituted translation system based on unmodified E. coli translation factors will be tested. Several methods for rapid peptide fragment isolation from the cell-free extract will be evaluated based on earlier work by AmberGen including: i) N-terminal epitope incorporation, ii) tRNA mediated protein engineering (TRAMPE) and iii) photocleavable biotin. Mass spectrometric analysis of the purified HIV peptides will be performed using MALDI-TOF mass spectrometry both on individual peptides and multiple peptides produced by multiplexing PCR and all downstream steps. Results will be compared to other methods of HIV retrovirus genotyping including DNA sequencing. In Phase II, a high throughput MS based system will be developed for HIV-1 drug-resistance testing based on the industry standard 96- or 384-well sample format. Dr. Richard D'Aquila, an internationally recognized expert on HIV-1 drug-resistance will serve as a consultant on this project and as a source of samples. Dr. Raymond Tellier, an infectious disease expert who has used mass spectrometry to characterize quasi-species variation in Hepatitis C Virus will also serve as a consultant.